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. 2016 Aug 3:9:62.
doi: 10.3389/fnmol.2016.00062. eCollection 2016.

Effects of Long-Term Environmental Enrichment on Anxiety, Memory, Hippocampal Plasticity and Overall Brain Gene Expression in C57BL6 Mice

Melanie Hüttenrauch  1 , Gabriela Salinas  2 , Oliver Wirths  1
Affiliations

Effects of Long-Term Environmental Enrichment on Anxiety, Memory, Hippocampal Plasticity and Overall Brain Gene Expression in C57BL6 Mice

Melanie Hüttenrauch et al. Front Mol Neurosci. .

Abstract

There is ample evidence that physical activity exerts positive effects on a variety of brain functions by facilitating neuroprotective processes and influencing neuroplasticity. Accordingly, numerous studies have shown that continuous exercise can successfully diminish or prevent the pathology of neurodegenerative diseases such as Alzheimer's disease in transgenic mouse models. However, the long-term effect of physical activity on brain health of aging wild-type (WT) mice has not yet been studied in detail. Here, we show that prolonged physical and cognitive stimulation, mediated by an enriched environment (EE) paradigm for a duration of 11 months, leads to reduced anxiety and improved spatial reference memory in C57BL6 WT mice. While the number of CA1 pyramidal neurons remained unchanged between standard housed (SH) and EE mice, the number of dentate gyrus (DG) neurons, as well as the CA1 and DG volume were significantly increased in EE mice. A whole-brain deep sequencing transcriptome analysis, carried out to better understand the molecular mechanisms underlying the observed effects, revealed an up-regulation of a variety of genes upon EE, mainly associated with synaptic plasticity and transcription regulation. The present findings corroborate the impact of continuous physical activity as a potential prospective route in the prevention of age-related cognitive decline and neurodegenerative disorders.

Keywords: Morris water maze; deep sequencing; dentate gyrus; enriched environment; hippocampal plasticity; neurogenesis; physical activity; stereology.

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Figures

FIGURE 1
FIGURE 1
Housing conditions and experimental design. (A) Exemplary pictures of standard (SH) and enriched (EE) housing conditions. Mice were housed in groups of 3–5. Enriched cages were equipped with three running wheels, tunnels, houses, and toys. (B) After weaning, C57Bl6/J mice were randomly allocated to either SH or EE conditions for 11 months. With 12 months of age, mice were tested in the open field and Morris water maze test followed by sacrifice and tissue collection.
FIGURE 2
FIGURE 2
Reduced anxiety upon enriched living conditions. (A) Enriched housed WT mice spent significantly more time in the center region of the open field compared to sedentary controls. The occupancy plots indicate exemplarily the running traces of SH and EE mice during the 5-min open field session. However, there was no difference in total time active (B), total distance traveled (C) and average speed (D) between standard and enriched C57Bl6/J mice. Unpaired t-test. ∗p < 0.05. All data were given as mean ± SEM (n = 7–10 per group).
FIGURE 3
FIGURE 3
Improved spatial memory performance in WT mice upon EE housing. (A) WT-SH and EE mice showed progressively decreasing escape latencies over the 3 days of cued training. (B) Similar to the cued training, SH- and EE-WT mice showed progressively reduced escape latencies over the 5 days of acquisition training. However, WT-EE mice displayed an improved spatial learning performance compared to their sedentary controls as seen by lower escape latencies over the whole training period. (C) WT-SH and EE mice displayed an intact spatial reference memory as they showed a clear and significant preference for the target (T) compared to all the other quadrants (L, R, O), with EE mice spending significantly more time in the target quadrant when compared to SH mice. The occupancy plots indicate the averaged swimming traces of standard and enriched housed mice during the probe trial. (D–F) No differences in swimming speed were observed between WT-SH and EE mice in cued training, acquisition training and probe trial. (A,B,D,E) Two-way repeated measures ANOVA. (C) One-way ANOVA followed by Bonferroni multiple comparisons. (F) Unpaired t-test. ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05. All data were given as mean ± SEM (n = 7–10 per group. L, left; R, right; O, opposite).
FIGURE 4
FIGURE 4
The effect of prolonged enrichment on hippocampal neuron numbers and hippocampal volume in WT mice. (A) Schematic representation of hippocampal counting areas. The CA1 region (outlined in green) was counted from Bregma -1.22 mm to -3.80 mm. The granule cell layer of the dentate gyrus (outlined in blue) was counted from Bregma -1.34 mm to -3.80 mm (modified from Paxinos and Franklin, 2001). (B) Design-based stereological analysis revealed no difference in CA1 neuron numbers between SH and EE mice. (C) The CA1 volume was significantly increased in WT mice housed under enriched living conditions when compared to sedentary controls (+29%). (D) Stereological analysis revealed a 15% increased dentate gyrus neuron number in EE compared to SH mice. (E) Analysis of dentate gyrus volume revealed a 36% increase in EE compared to SH mice. (B–E) Unpaired t-test. ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05. All data were given as mean ± standard deviation (SD); (n = 6–7 per group).
FIGURE 5
FIGURE 5
Enriched environment had no effect on adult neurogenesis in 12-month-old WT mice. (A,B) New-born doublecortin (DCX)-positive neurons were stained and quantified in the subgranular zone (SGZ) of the dentate gyrus. Adult neurogenesis was found to be unchanged upon long-term enriched living conditions in C57Bl6/J mice. Unpaired t-test. All data were given as mean ± standard deviation (SD); (n = 7 per group). Scale bar: 100 μm.
FIGURE 6
FIGURE 6
Gene expression profile changes upon long-term EE in C57Bl6/J mice. (A) Heatmap of differentially expressed genes (DEGs) between SH and EE WT mice. Each column represents a pooled sample of two brain hemispheres (A1-3 = SH, B1-3 = EE) and each row represents the top genes that were differentially expressed. Yellow indicates upregulated genes, blue indicates downregulated genes. (B) Volcano plot of showing up- (red) or down-regulated (blue) genes in 12-month-old WT animals upon long-term enriched housing. (C) Deep sequencing data were validated using qRT-PCR analysis. Normalization was performed against the house-keeping gene actin. Unpaired t-test. ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05. All data were given as mean ± SEM (n = 6 per group).
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